Genome walking refers to a methodology used to clone unknown genomic regions adjacent to known sequences (“known region”), and is also called chromosome walking.
Until now, many methods have been developed for genomic walking. The traditional approach consists of the construction and screening of genomic DNA libraries using the known base sequence as a probe. Before the development of Polymerase Chain Reaction (PCR), this method was most widely used, but has disadvantages in that libraries should be constructed and a great deal of manpower and time are required during the screening process.
The most commonly used PCR based genome walking method is Inverse PCR, where genomic DNA fragments are ligated into circles and the primers designed to recognize the known region in a reverse direction are used to perform amplification (Triglia et al., Nucleic Acid Res. 1988, 16, 8186). One of the drawbacks of Inverse PCR is the requirement for suitable restriction enzyme sites that flank the priming region.
Another method is vectorette PCR, where genomic DNA treated with restriction enzymes is ligated to a cassette containing a mismatch region and then PCR is performed (Riley et al., Nucleic Acid Res. 1990, 18, 2887), and various types of linker- or adapter-ligated PCR have been developed (Isegawa et al., Mol. Cell. Probe. 1992, 6, 467; Siebert et al., Nucleic Acid Res. 1995, 23, 1087; Kilstrup and Kristialsen, Nucleic Acid Res. 2000, 28, e55; Nthangeni et al., J. Microbiol. Methods. 2005, 61, 225).
To improve these methods, methods of performing secondary PCR using blocked adapters (Padegimas and Reichert, Anal. Biochem 1998, 260, 149; Reddy et al., Mol. Biotechnol. 2002, 22, 223), biotinylated primers (Rosenthal and Jones, Nucleic Acid Res. 1990, 18, 3095) or nested primers were developed (Rosenthal, Trends. Biotechnol. 1992, 10, 44). In addition, a method of cloning desired regions using random primers without cassette ligation has been developed (Parker et al., Nucleic Acid Res. 1991, 19, 3055; Park et al., Nucleic Acid Res. 1991, 19, 7155; Domingue and Lopez-Larrea, Nucleic Acid Res. 1994, 22, 3247; Liu and Whittier, Genomics, 1995, 25, 674; Trueba and Johnson, Biotechniques, 1996, 21, 20; Tamme et al., Biotechniques, 2000, 28, 895; Karlyshev et al., Biotechniques, 2000, 28, 1078).
As such, many studies have been made to improve the genome walking method, and thus significant progress has been made. However, it is still in need of improvement. The most serious problems of the PCR-based genome walking methods are low amplification efficiency and non-specific amplification. That is, unwanted genes are amplified together, which makes it difficult to distinguish amplification of desired genes. To improve this problem, complicated multi-step methods were developed, including secondary PCR, purification of desired PCR products using biotinylated primers, or improvement of PCR cycle conditions. However, satisfactory performance has not yet been achieved.